Methods to covalently attach labels and reporter molecules to oligonucleotides have provided valuable tools in the field of molecular biology and gene probe diagnostics. Recent technologies in non-isotopic gene probes, DNA sequencing (Connell, C. et al. [1987] Biotechniques 5:342-346; Kaiser, R., S. Mackellar, R. Vinayak, J. Sanders, R. Saavedra, L. Hood [1989] Nucleic Acids Res. 17:6087-6102), electron microscopy (Sproat, B. S., B. Beijer, P. Rider [1987] Nucleic Acids Res. 15:6181-6196), and X-ray crystallography (Sproat et al. [1987] Nucleic Acids Res. 15:4837-4848) have provided impetus for the development and improvement of such methods. New and emerging applications employing the polymerase chain reaction (PCR) (Hultman, T., S. Bergh, T. Moks, M. Uhlen [1991] Biotechniques 10:84-93; Landgraf, A., B. Reckmann, A. Pingoud [1991] Analytical Biochemistry 193:231-235; Zimran, A., C. Glass, V. Thorpe, E. Beutler [1989] Nucleic Acids Res. 17:7538) have further expanded the need for convenient and versatile reagents to chemically modify oligonucleotides.
Current methods to introduce chemical modifications into oligonucleotides employ special non-nucleosidic phosphoramidite reagents during automate oligonucleotide synthesis. The methods are limited to single modifications at only the 5' terminus. The inherent disadvantage of such methods is that the reagents terminate chain elongation at the point they are introduced (5' terminus) and therefore only single modifications can be performed. Chemical modifications that have been introduced in this fashion are primary aliphatic amine (Sinha, N. D., R. M. Cook [1988] Nucleic Acids Res. 16:2659-2669) and thiol (Connolly, B. [1985] Nucleic Acids Res. 13:4485-4502) functionalities. Oligonucleotides functionalized with primary aliphatic amines or thiol groups must be subsequently derivatized with labels such as biotin, fluorescein, and enzymes. Subsequent derivatization requires a second reaction and purification step which minimizes the convenience and practicality of this method. Cocuzza expanded this method to directly incorporate a single biotin label into an oligonucleotide at the 5' terminus (Cocuzza, A. [1989] Tetrahedron Lett. 30:6287-6290).
Recently, Nelson et al. introduced a new type of non-nucleosidic phosphoramidite reagent that utilized a 1,2-ethanediol backbone (Nelson, P., R. Sherman-Gold, R. Leon [1989] Nucleic Acids Res. 17:7179-7186). This reagent allowed primary aliphatic amines to be incorporated multiple times and at any position of the oligonucleotide. The development of this method eliminated the termination of chain elongation during synthesis, an inherent problem of the above method. Employment of the 1,2-ethanediol backbone allowed the phosphoramidite reagent to be incorporated exactly like a normal nucleoside phosphoramidite, at any position and multiple times. Misiura et al. expanded the use of the 1,2-ethanediol backbone derived from a glycerol intermediate, to directly incorporate multiple biotins into oligonucleotides (Misiura, K., I. Durrant, M. Evans, M. Gait [1990] Nucleic Acids Res. 18:4345-4354). The development of the 1,2-ethanediol backbone modification method provided better utility and versatility, especially in the field of gene probe diagnostics where multiple labels yield greater signal detection.
There still remain some serious disadvantages in the use of 1,2-ethanediol phosphoramidite modification reagents. First, when internally incorporated into an oligonucleotide, the internucleotide phosphate distance is constricted and is one carbon atom short of the natural 3-carbon atom internucleotide distance. This directly affects hybridization and annealing properties, resulting in destabilization. Secondly, the spacer arm connected to the 1,2-ethanediol backbone is very short (1-4 atoms) Attachment of labels and reporter molecules to the spacer arm can result in steric hindrance being too close to the oligonucleotide. This is an important factor for antibody binding and signal detection. Lastly, the chemistry to modify spacer arm length and to attach different functional groups and labels to the 1,2-propanediol backbone is limited and difficult. Another procedure by Zuckerman et al. (Zuckerman, R., D. Corey, P. Schulz [1987] Nucl. Acids Res. 5305-5321) incorporates a 3' terminal thiol group via solid phase oligonucleotide synthesis. Although this procedure has some advantages, it requires many synthetic steps and purifications.
The purpose of the present invention is to overcome the disadvantages encountered in the prior art by providing improved non-nucleosidic reagents to directly modify or label oligonucleotides via automated solid phase synthesis. Also provided is a unique, simple, and versatile synthesis strategy for modifying spare arm length and attaching different functional groups and labels when preparing such reagents.